Use of AP-1 activators to treat glaucoma and ocular hypertension

ABSTRACT

Compositions comprising AP-1 activators and methods of use for treating glaucoma and ocular hypertension are disclosed.

This application claims the benefit of Provisional Application No.60/130,133, filed Apr. 20, 1999.

The present invention relates to the treatment of glaucoma and ocularhypertension. In particular, the present invention relates to the use ofcompounds that increase the activity of Activator Protein-1 (AP-1) totreat glaucoma and ocular hypertension.

BACKGROUND OF THE INVENTION

Glaucoma is a progressive disease which leads to optic nerve damage and,ultimately, total loss of vision. The causes of this disease have beenthe subject of extensive studies for many years, but are still not fullyunderstood. The principal symptom of, and/or risk factor for, thedisease is elevated intraocular pressure (IOP) or ocular hypertension.

The reasons why IOP is increased in glaucoma patients are not fullyunderstood. It is known that elevated IOP can be at least partiallycontrolled by administering drugs which either reduce the production ofaqueous humor within the eye, such as beta-blockers and carbonicanhydrase inhibitors, or increase the flow of aqueous humor out of theeye, such as cholinergic agonists and sympathomimetics.

All types of drugs currently being used to treat glaucoma havepotentially serious side effects. Cholinergic agonists such aspilocarpine can cause blurring of vision and other ocular side effects,which may lead either to decreased patient compliance or to terminationof therapy. Systemically administered carbonic anhydrase inhibitors canalso cause serious side effects, such as nausea, dyspepsia, fatigue, andmetabolic acidosis which can affect patient compliance and/ornecessitate the withdrawal of treatment. Moreover, some beta-blockershave been known to be associated with pulmonary side effectsattributable to their effects on beta-2 receptors in the pulmonarytissue. Sympathomimetics cause tachycardia, arrhythmia and hypertension.There is therefore a continuing need for new therapies which controlelevated IOP associated with glaucoma.

Activator protein-1 (AP-1) is a dimeric gene transcription promotercomprised of subunit proteins which are the products of at least threedifferent proto-oncogene families: the Jun (c-Jun, v-Jun, JunB, JunD),Fos (c-Fos, v-Fos, FosB, FosB2, Fra-1, Fra-2) or activatingtranscription factor (B-ATF, ATF2, ATF3/LRF1) families. Uncomplexedmonomers and homo- and hetero-dimers of these protein subunits have beenobserved in a variety of mammalian and non-mammalian tissues (Foletta etal., Transcriptional regulation in the immune systems: all roads lead toAP-1, J. Leukoc Biol. volume 63, pages 139–152 (1998); and Karin et al.,AP-1 function and regulation, Curr. Opin. Cell Biol., volume 9, pages240–246 (1997)).

AP-1 binds to specific DNA sequences within enhancer regions of manygenes (e.g., TPA (12-O-tetradecanoylphorbol-13-acetate) responseelements (TREs) or cyclic AMP response elements (CREs)) and promotes theactivation of the particular gene. Examples of genes which contain AP-1consensus seqences in their enhancer regions include the genes for SV40and human metallothionein IIA (Lee et al., Activation of transcriptionby two factors that bind promoter and enhancer sequences of the humanmetallothionein gene and SV40, Nature, volume 325, pages 368–372). Thebinding affinity of AP-1 for the various response elements appears todepend on the specific protein subunit dimer complex. Dimers composed ofJun/Jun or Jun/Fos generally bind TREs, whereas ATF/ATF or Jun/ATFdimers preferentially bind CREs (Whitmarsh et al., Transcription factorAP-1 regulation by mitogen-activated protein kinase signal transductionpathways, J. Mol. Med., volume 74, pages 589–607 (1996); and Karin etal., Current Opin. Cell Biol., volume 9, pages 240–246 (1997)). Thebiological consequences of AP-1 mediated gene transcription may alsovary, depending upon the dimer composition. For example, induction ofmurine glutathione-S-transferase genes is apparently mediated by aFos/Jun heterodimer which binds at least one TRE sequence within thegene's antioxidant response element (Ainbinder et al., Regulatorymechanisms involved in activator-protein-1 (AP-1)-mediated activation ofglutathione-S-transferase gene expression by chemical agents, Eur. J.Biochem., volume 243, pages 49–57 (1997); Xie et al., ARE-andTRE-mediated regulation of gene expression, J. Biol. Chem., volume 270,pages 6894–6900 (1995)), and a c-Jun/ATF-2 complex has been shown tobind to a CRE involved in activation of the T-cell gene for tumornecrosis factor-alpha (Foletta et al., J. Leukoc. Biol., volume 63,pages 139–152 (1998)).

Several AP-1 Activators have been reported previously, includingβ-naphthoflavone and tert-butylhydroquinone (tBHQ) (Ainbinder et al.,Eur. J. Biochem., volume 243, pages 49–57 (1997); Ainbinder et al.,Signaling pathways in the induction of c-fos and c-jun proto-oncogenesby 3-methylcholanthrene, Receptors and Signal Transduction, volume 7,pages 279–289 (1998); and Oazki et al., The comparative effects ofhaloperidol, (−)-sulpiride, and SCH23390 on c-fos and c-jun mRNAexpressions, and AP-1 DNA binding activity, Eur. Neuropsychopharmacol.,volume 7, pages 181–187 (1997)). Nowhere in the art, however, has itbeen taught or suggested that AP-1 activators may be useful in treatingglaucoma or ocular hypertension.

SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods of use forthe treatment of glaucoma and ocular hypertension. More specifically,the invention is directed to compositions comprising AP-1 activators andmethods of use.

Preferred methods involve the topical administration of compositionscomprising tBHQ.

DESCRIPTION OF THE DRAWING

FIG. 1 is a graph showing the effects of tBHQ on IOP in a human ocularperfusion organ culture model.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compositions and methods of use forthe treatment of glaucoma and ocular hypertension.

The compounds useful in the present invention compositions and methodsare AP-1 Activators. While not intending to be bound by any theory, itis believed that the stimulation of AP-1 activity leads to thetranscription of mRNAs and corresponding translation of proteins usefulin effecting the maintenance or lowering of IOP. As used herein, “AP-1Activator” refers to those molecules which lower IOP via AP-1 mediatedtranscription. Such activators may include agents which (1) increase theformation and expression of proteins comprising AP-1 complex(es), (2)enhance the formation of such complexes, or (3) promote binding of anAP-1 complex to regulatory/promoter sites on cellular genes. Examples ofAP-1 Activators include β-naphthoflavone, tert-butylhydroquinone (tBHQ),sulpiride, haloperidol, and 3-methylcholanthrene. The most preferredAP-1 Activator is tBHQ.

Other AP-1 Activators may be elucidated by methods known in the art. Forexample, an increase in the level of DNA-bound AP-1 complex, as assessedby electrophoretic mobility shift assay, is an accepted means fordetection of activated AP-1 activity. Such DNA-binding can be determinedby the following protocol:

DNA-Binding Assay:

Target cellular tissue (e.g., human trabecular meshwork tissue and/orcultured cells) is treated with or without an AP-1 Activatorcandidate(s). Nuclear extracts of the cells are then prepared accordingto the method of Schreiber et al., Rapid detection of octamer bindingproteins with ‘mini-extracts’ prepared from a small number of cells,Nucleic Acids Res., volume 17, page 6419 (1989). Electrophoreticmobility shift assays are then carried out with the nuclear extracts asdescribed by Ainbinder et al., Regulatory mechanisms involved inactivator-protein-1 (AP-1)-mediated activation ofglutathione-S-transferase gene expression by chemical agents, Eur. J.Biochem., volume 243, pages 49–57 (1997). Briefly, extracts areincubated with a ³²P-labelled AP-1 oligonucleotide probe and theresulting complexes are then separated by non-denaturing acrylamide gelelectrophoresis. Levels of DNA binding activity can then be determinedfrom analysis of autoradiograms of the dried gels. By using this method,compounds such as tBHQ have been demonstrated to be AP-1 Activators.

Other AP-1 Activators may be identified by routine methods known in theart. For example, methods disclosed in the following publications may beuseful in elucidating other AP-1 Activators of the present invention:

-   -   (1) Ainbinder et al., Regulatory mechanisms involved in        activator-protein-1 (AP-1)-mediated activation of        glutathione-S-transferase gene expression by chemical agents,        Eur. J. Biochem., volume 243, pages 49–57 (1997);    -   (2) Ainbinder et al., Signaling pathways in the induction of        c-fos and c-jun proto-oncogenes by 3-methylcholanthrene,        Receptors and Signal Transduction, volume 7, pages 279–289        (1998);    -   (3) Ozaki et al., The comparative effects of haloperidol,        (−)-sulpiride, and SCH23390 on c-fos and c-jun mRNA expressions,        and AP-1 DNA binding activity, Eur. Neuropsychopharmacol.,        volume 7, pages 181–187 (1997); and    -   (4) Xie et al., ARE-and TRE-mediated regulation of gene        expression, J. Biol. Chem., volume 270, pages 6894–6900 (1995);        the foregoing publications are incorporated herein by reference.

EXAMPLE 1

The following example illustrates the effect of an AP-1 Activator, tBHQ,on aqueous outflow facility as demonstrated using a human ocularperfusion organ culture model. Pharmaceutical candidates which maintainor increase outflow facility are believed to be useful in treating orcontrolling IOP and, hence, glaucoma. The study was performed asfollows:

Human donor eyes less than 24 hours post mortem were used. The globe wassectioned equatorially followed by the removal of vitreous, lens,zonules, iris, and most of the ciliary body. The anterior segment wasthen mounted on a perfusion dish, perfused with cell culture medium at aconstant hydrostatic pressure of 10 mm Hg (Johnson et al., Humantrabecular meshwork organ culture: A new method, Invest Ophthalmol VisSci., volume 28, pages 945–953 (1987); Erickson-Lamy et al., Outflowfacility studies in the perfused human ocular anterior segment, Exp. EyeRes., volume 52, pages 723–731 (1991); and Clark et al.,Dexamethasone-induced ocular hypertension in perfusion cultured humaneyes, Invest. Ophthalmol. Vis. Sci., volume 36, pages 478–489 (1995)).The flow rate of the perfusate was measured by weighing the reservoir atpredetermined periods. After 2 to 4 days of equilibrium period, one eyeof each of the donor pair was perfused with the test compound, t-BHQ (10μM), while the other was perfused with vehicle (control). The resultsare shown in FIG. 1 and Table 1, below:

TABLE 1 Effect of t-BHQ (10 μM) on Aqueous Outflow Rate in Human OcularPerfusion Organ Culture Flow Rate (% of Time 0) Time after Treatment(Mean ± SEM, n = 6) (Hours) Vehicle t-BHQ −4 100.0 ± 0.8   97.0 ± 4.0 0100.0 100.0 4 92.0 ± 3.6 105.8 ± 5.4* 24 92.0 ± 4.8 102.9 ± 3.9* 48 86.6± 9.2 105.2 ± 10.1 72  76.6 ± 12.5 111.8 ± 11.8* 96  61.8 ± 12.1 120.9 ±16.4* Note: (1) The basal flow rate at time 0 for vehicle-treated eyeswas 3.55 ± 1.00 μL/min, and that for drug-treated eyes was 4.09 ± 0.63μL/min. (2) *represents p < 0.05, paired t-test compared to thevehicle-treated eye of the same time point.

While not intending to be bound by any theory, it is believed that theAP-1 Activators of the present invention are useful in increasing theoutflow facility of a mammal's eye, which leads to maintained or loweredIOP, and thus useful in the treatment of glaucoma or ocularhypertension.

The preferred route of administration for the methods of the presentinvention is topical. The preparation of topical ophthalmic compositionsis well known in the art. Generally, topical ophthalmic compositionsuseful in the present invention will be in the form of a solution,suspension, gel, or formulated as part of a device, such as a collagenshield or other bioerodible or non-bioerodible device. Variousexcipients may be contained in the topical ophthalmic solutions,suspensions or gels of the present invention. For example, buffers(e.g., borate, carbonate, phosphate), tonicity agents (e.g., sodiumchloride, potassium chloride, polyols), preservatives (e.g.,polyquaterniums, polybiguanides, benzalkonium chloride), chelatingagents (e.g., EDTA), viscosity enhancing agents (e.g., polyethoxylatedglycols) and solubilizing agents (e.g., polyethoxylated castor oils,including polyoxl-35 castor oil (Cremophor EL®, BASF Corp., Parsippany,N.J.); Polysorbate 20, 60 and 80; Pluronic® F-68, F-84 and P-103 (BASFCorp.); or cyclodextrin) may be included in the topical ophthalmiccompositions. However, preferable compositions of the present inventionwill not include preservatives or tonicity agents which are known toadversely affect or irritate the eye.

A variety of gels may be useful in topical ophthalmic gel compositionsof the present invention, including, but not limited to, carbomers,polyvinyl alcohol-borates complexes, or xanthan, gellan, or guar gums.Topical ophthalmic bioerodible and non-bioerodible devices are known inthe art and may be useful in the topical administration of the AP-1Activators. See, for example, Weiner, A. L., Polymeric Drug DeliverySystems For the Eye, in Polymeric Site-specific Pharmacotherapy, Ed., A.J. Domb, John Wiley & Sons, pages 316–327 (1994). While the particularingredients and amounts to be contained in topical ophthalmiccompositions useful in the methods of the present invention will vary,particular topical ophthalmic compositions will be formulated to effectthe administration of one or more AP-1 Activators topically to the eye.

In general, the doses of AP-1 Activators used for the above describedpurposes will vary, but will be in an amount effective to maintain orlower IOP, or otherwise treat glaucoma or ocular hypertension. As usedherein, the term “pharmaceutically effective amount” refers to thatamount of an AP-1 Activator administered to a mammal which maintains orlowers IOP, or otherwise ameliorates the glaucomatous condition of thepatient. The AP-1 Activators will normally be contained in thecompositions described herein in an amount from about 0.00001 to about2.0 percent weight/volume (“% w/v”), preferably 0.01 to 2% w/v. Thecompositions of the present invention may be delivered topically to theeye, about one to six times a day.

As used herein, the term “pharmaceutically acceptable vehicle” refers toany formulation which is safe, and provides the appropriate delivery forthe desired route of administration of an effective amount of at leastone AP-1 Activator of the present invention.

Preferred formulations of AP-1 Activators combinations of the presentinvention include the following Examples 2–3:

EXAMPLE 2

Topical Ophthalmic Formulation:

Ingredient Amount (% wt) AP-1 Activator 0.01 to 2 Phosphate BufferedSaline 1.0 Benzalkonium Chloride 0.01 Polysorbate 80 0.5 Purified waterq.s. to 100%

EXAMPLE 3

Topical Ophthalmic Formulation:

Ingredient Amount (% wt) t-HBQ 0.01 to 2 Phosphate Buffered Saline 1.0Hydroxypropyl-β-cyclodextrin 4.0 Purified water q.s. to 100%

EXAMPLE 4

Preferred Formulation for a Topical Ophthalmic Solution:

Component Amount (% wt) t-HBQ 0.01 to 2 Benzalkonium chloride 0.01 HPMC0.5 Sodium chloride 0.8 Sodium phosphate 0.28 Edetate disodium 0.01NaOH/HCl q.s. to pH 7.2 Purified Water q.s. to 100%

1. A method for treating glaucoma or ocular hypertension which comprisesadministering to a mammal a composition comprising an AP-1 Activatorselected from the group consisting of β-naphthoflavone,3-methylcholanthrene, and tert-butylhydroquinone, and a pharmaceuticallyacceptable vehicle.
 2. A method according to claim 1, wherein the AP-1Activator is tert-butylhydroquinone.
 3. A method according to claim 1,wherein the composition comprises: about 0.01 to 2.0% w/v t-BHQ about0.28% w/v sodium phosphate; about 0.8% w/v sodium chloride; about 0.01%w/v benzalkonium chloride about 0.5% w/v HPMC about 0.1% w/v edetatedisodium; and water.